灵芝疏水蛋白编码基因Unigene0004454的克隆和异源表达

Cloning and Heterologous Expression of Hydrophobin Encoding gene Unigene0004454 of Ganoderma lucidum

摘要:

为探究灵芝（Ganoderma lucidum）ZJ-1菌株疏水蛋白编码基因Unigene0004454的潜在功能，该研究通过基因克隆、生物信息学分析、启动子元件分析和基因异源表达对该基因展开研究。结果表明，Unigene0004454的DNA序列长度为436 bp，cDNA序列长度为330 bp，编码109个氨基酸。氨基酸序列分析结果表明其具有疏水蛋白典型的半胱氨酸结构特点；在进化上与白肉灵芝（Ganoderma leucocontextum）、紫芝（Ganoderma sinense）的疏水蛋白亲缘关系较近。Unigene0004454的启动子区域含有核心启动子元件（如TATA-BOX）、多个激素响应元件（如ABRE）及环境胁迫响应元件（如MBS），推测Unigene0004454的生理功能可能与菌株生长发育有关。构建pET-32a-Unigene0004454重组质粒并转入大肠杆菌BL21（DE3）感受态细胞进行表达，经SDS-PAGE和Western blotting验证，结果表明在BL21菌株中成功表达了分子量约为27 kDa的重组蛋白，验证了Unigene0004454基因异源表达的可行性。该研究为进一步阐明Unigene0004454基因的生理功能及开发疏水蛋白的应用奠定了基础。

Abstract:

To investigate the potential functions of Unigene0004454 gene, which encodes a hydrophobin in Ganoderma lucidum ZJ-1 strain, this study adopted approaches involving gene cloning, bioinformatics analysis, promoter element analysis, and heterologous expression experiment. The results indicated that the full length of Unigene0004454 gene was 436 bp, a cDNA length 330 bp, and encoded 109 amino acids. Amino acid sequence analysis demonstrated that it has the typical cysteine structure characteristic of hydrophobins, and showed a closer evolutionary relationship with hydrophobins from Ganoderma leucocontextum and Ganoderma sinense. The promoter region of Unigene0004454 contains core promoter element (such as TATA-BOX), multiple hormone responsive elements (such as ABRE), and environmental stress inducible elements (such as MBS). It is speculated that the physiological function of Unigene0004454 may be related to the growth and development of the strain. The recombinant plasmid pET-32a-Unigene0004454 was constructed and transferred into E. coli BL21(DE3) competent cells for expression, followed by SDS-PAGE and Western blotting validation. The results indicated that the recombinant protein with a molecular weight of approximately 27 kDa was successfully expressed in BL21 strain, thereby verifying the feasibility of heterologous expression for Unigene0004454. This study lays a foundation for further elucidation of the physiological functions of Unigene0004454 gene and the development of hydrophobin-based applications.